Synthesis of tryptophan-dehydrobutyrine diketopiperazine and biological activity of hangtaimycin and its co-metabolites

An improved synthesis for tryptophan-dehydrobutyrine diketopiperazine (TDD), a co-metabolite of the hybrid polyketide/non-ribosomal peptide hangtaimycin, starting from ʟ-tryptophan is presented. Comparison to TDD isolated from the hangtaimycin producer Streptomyces spectabilis confirmed its S configuration. The X-ray structure of the racemate shows an interesting dimerisation through hydrogen bridges. The results from bioactivity testings of hangtaimycin, TDD and the hangtaimycin degradation product HTM222 are given.


S4
To a solution of L-threonine (S1) (2.00 g, 16.8 mmol) in H2O (34 mL) was added NaHCO3 (2.17 g, 25.9 mmol), followed by (Boc)2O (5.72 g, 26.2 mmol) dissolved in MeOH (34 mL). After the reaction mixture was stirred at room temperature for 24 h, MeOH was removed in vacuum. The residue was acidified with aq. HCl solution (2 N) until the pH = 2 was reached. The aqueous phase then was extracted with EtOAc (3 × 100 mL). The combined organic phases were dried with MgSO4 and concentrated under reduced pressure to give S2 as colorless oil. Compound S2 (1.10 g, 5.0 mmol) was added to a solution of 8 (1.16 g, 5.0 mmol) and N,N-diisopropylethylamine (2.09 mL, 12.0 mmol) in CH2Cl2 (40 mL). After the mixture was cooled to 0 °C BOP-Cl (1.40 g, 5.5 mmol) was added. The reaction solution was stirred at room temperature for 15 h and then diluted with EtOAc (200 mL). After washing with sat. aq. NH4Cl solution (30 mL), sat. aq. NaHCO3 solution (30 mL) and brine (30 mL), the organic phase was dried with MgSO4 and concentrated in vacuum.
The residue was purified by column chromotography on silica gel (EtOAc) to give 9 as yellowish oil.
Methyl N  -((tert-butoxycarbonyl)-L-threonyl)-N  -methyl-L-tryptophanate (9  To a solution of 9 (85 mg, 0.20 mmol) in CH2Cl2 (1 mL) was added trifluoroacetic acid (0.05 mL). After stirring at room temperature for 30 min, the reaction mixture was concentrated under reduced pressure. Then another portion of CH2Cl2 (1 mL) was added to the residue, followed by triethylamine (40 mg, 0.40 mmol). After stirring at room temperature for 2 h, the reaction mixture was concentrated under reduced pressure again. The residue was then purified by column chromotography on silica gel (EtOAc/MeOH = 6:1) to give 10 as yellowish oil. NaHCO3 solution (30 mL) and brine (30 mL). The organic layer was then dried with MgSO4 and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (cyclohexane/EtOAc = 5:1) to give 11 as colorless oil.    After washing with sat. aq. NH4Cl solution (30 mL), sat. aq. NaHCO3 solution (30 mL) S11 and brine (30 mL), the organic phase was dried with MgSO4 and concentrated in vacuo.

Single crystal X-ray diffraction study
Single crystals of (rac)-4 were grown from a concentrated solution in methanol upon standing at −20 °C. The data collection of a suitable clear colourless plate-like crystal was perfomed on a Bruker D8 Venture diffractometer using Cu-Kα radiation (λ = 1.54178 Å). The diffractometer was equipped with a low-temperature device (Cryostream 800er series, Oxford Cryosystems, 100(2) K). Intensities were measured by fine-slicing ω-and -scans and corrected for background, polarisation and Lorentz effects. An empirical absorption correction was applied for the data set following Blessing's method [2]. The structure was solved by intrinsic phasing methods [3] and refined anisotropically by the least-squares procedure implemented in the XL program system [4]. The carbon-bonded hydrogen atoms were included isotropically using the riding model on the bound carbon atoms, the positions of hydrogen atoms at hetero atoms (O, N) were refined freely. CCDC 2182500 contains the supplementary crystallographic data for this paper, which can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. S15

Determination of the minimal inhibitory concentration (MIC)
The minimal inhibitory concentration (MIC) was determined in cation-adjusted Mueller-Hinton medium (MH II) that contains casein, beef extract and starch by using a twofold serial dilution method according to the standards and guidelines of the Clinical and Laboratory Standards Institute (CLSI) [5]. Due to solubility issues of some of the compounds at high concentrations, a predilution of all compounds was made in DMSO.
The test compounds were dissolved in DMSO at 20 mg/mL and a two-fold serial dilution was prepared in round-bottom polystyrene microtiter plates (Sarstedt, Germany) in  [8], Escherichia coli HN817 and E. coli HN818 [9,10] were used as further reference strains. The ATCC strains were provided by the American Type Culture Collection. A. baumannii 09987 was obtained from the University of Bonn, Germany.

Growth curves (MIC)
For continuous recording growth curves, samples were prepared as above with the exception that flat-bottom polystyrene plates (Sarstedt, Germany) were used and that the plates were incubated at 37 °C in a Tecan SPARK microplate reader overnight with intermittent shaking for 30s every 10 min. The optical density at 600 nm was recorded directly after each shaking interval. The medium background of the sterile control was subtracted from the OD600 values of the bacterial cultures. Means and standard deviations were calculated using GraphPad Prism.